Biochemical and molecular biological studies on animal lectins

动物凝集素的生化和分子生物学研究

• 批准号: 04680172
• 负责人: KASAI Ken-ichi
• 金额: $ 1.22万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680172/
• 关键词: lectin; galactoside; nematode; C.elegans; glycoconjugate; carbohycrate-binding protein; C.egegans

This research project was aimed to elucidate biological significance of not only animal metal-independent beta-galactoside-binding lectin (galectin) but also complex carbohydrates. Vertebrate galectins (human and chicken) and nematode galectin (C.elegans) were studied from a variety of viewpoints. A procedure which enables large-scale production of a recombinant human galectin in E.coli was established. This also enabled to produce a number of mutant lectins by site-directed mutagenesis, and roles of conserved amino acid residues were clarified. One of the mutants proved to be extremely useful because it acquired significant stability which promise success of a variety of experiments which had been difficult because of the instability of the natural galectin. The role of galectin in morphogenesis of the skin during the development of chick embryo was studied in detail by immunohistochemical technique. The nematode galectin is the first galectin found in invertebrates. We determined its complete primary structure, cloned its cDNA and established a production system of its recombinant protein in E.coli. The nematode galectin proved to be composed of tandemly repeated two homologous domains. Production of each half molecule by gene engineering revealed that both have saccharide-binding ability. Distribution of the galectin during embryogenesis of C.elegans was also studied by immunohistochemistry, and interesting observations are now accumulating.

本研究旨在阐明动物金属非依赖性β-半乳糖苷结合凝集素(Galectin)以及复合碳水化合物的生物学意义。从不同角度研究了脊椎动物Galectin(人和鸡)和线虫Galectin(C.elegans)。建立了一种在大肠杆菌中大规模生产重组人半乳糖凝集素的方法。这也使得通过定点突变产生了许多突变的凝集素，并阐明了保守氨基酸残基的作用。其中一个突变体被证明是非常有用的，因为它获得了显著的稳定性，这保证了各种实验的成功，这些实验由于天然半乳糖凝集素的不稳定而变得困难。应用免疫组织化学技术，详细研究了半乳糖凝集素在鸡胚发育过程中对皮肤形态发生的影响。线虫Galectin是第一种在无脊椎动物中发现的Galectin。我们确定了其完整的一级结构，克隆了其cDNA，并建立了其重组蛋白在大肠杆菌中的表达体系。半乳糖凝集素是由两个重复的同源结构域组成的。通过基因工程生产的每半个分子都具有与糖结合的能力。用免疫组织化学方法研究了半乳糖凝集素在线虫胚胎发育过程中的分布，目前正在积累有趣的观察结果。

项目成果

Hirabayashi,J.: "Evidence that Caenorhabditis elegans 32-kDa β-galactoside-binding protein is homologous to vertebrate β-galactoside-binding lectins." J.Biol.Chem.267. 15485-15490 (1992)

Hirabayashi, J.：“秀丽隐杆线虫 32-kDa β-半乳糖苷结合蛋白与脊椎动物 β-半乳糖苷结合凝集素同源的证据。J.Biol.Chem.267 (1992)。

Hirabayashi, J.: "Affinity tag procedure using anhydrotrypsin agarose." J.Chromatogr.597. 181-187 (1992)

Hirabayashi, J.：“使用脱水胰蛋白酶琼脂糖的亲和标签程序。”

Hirabayashi,J.: "The family of metazoan beta-galactoside-binding lectins:Structure,function and molecular evolution." Glycobiology,. 3. 297-304 (1993)

Hirabayashi, J.：“后生动物β-半乳糖苷结合凝集素家族：结构、功能和分子进化。”

Akimoto Y.: "Changes in expression of the endogeneous beta-galactoside-binding 14-kDa lectin of chick embryonic skin during epidermal differentiation." Exp.Cell Res.199. 297-305 (1992)

Akimoto Y.：“表皮分化过程中鸡胚皮肤内源性 β-半乳糖苷结合 14-kDa 凝集素表达的变化。”

Hirabayashi, J.: Gabius, H., (Editor) Lectins and Glycobiology Springer, Berlin. Construction of mutant lectin genes by oligonucleotide-directed mutagenesis and their expression in E.coli., 482-491 (1993)

Hirabayashi, J.：Gabius, H.，（编辑）凝集素和糖生物学 Springer，柏林。